The focus of this research proposal is the determination of molecular and catalytic properties of four members of the CYP4A gene subfamily of cytochromes P450 which hydroxylate fatty acids and eicosanoids primarily or exclusively in the psi-position. The original member of this closely related (greater than 85% homologous at the amino acid level) subfamily was the P4504A4, simultaneously isolated in this laboratory from the lungs of pregnant rabbits and from Kusunose's laboratory from progesterone-treated male rabbits. Johnson, et al cloned and expressed three kidney cDNAs encoding lauric acid psi-hydroxylases now known as CYP 4A5, 4A6, and 4A7. A partial cDNA encoding CYP4A4 was reported by Kusunose's laboratory and the intact, full length cDNA was isolated jointly by Johnson's and masters' laboratories. Although the functions of these cytochromes P450 are unknown, a large literature is developing in which they are implicated in hemodynamic function by controlling vascular tone. Cerebral and renal microvessels are contracted by 20- hydroxyeicosatetraenoic acid (the psi-hydroxylated product of arachidonic acid) at concentrations of less than 10 -10M. Due to the high degree of sequence homology of these enzymes found in kidney and lung, their implication in hemodynamic control, and their degree of substrate specificity, the following Specific Aims are planned: 1) Expression of the CYP4A7 gene product will be pursued using other E. coli strains as well as other vectors, in the presence and absence of a plasmid encoding E. coli chaperonins (groEL and groES). 2) The reconstitution of these cytochromes P450 will be attempted using conventional sonicated lipid preparations and by incorporation into liposomes (lipid vesicles) prepared by various techniques, including the use of amphipathic detergents. 3) site-directed mutagenesis based upon identifying specificity determinants from chimeric constructs will be performed with each of the CYP4A enzymes (4A4-4A7) and mutants will be expressed in E. coli. 4) By homology-based modeling of the CYP4A enzymes using the Bacillus megaterium heme domain crystal structure, modules and regions of these enzymes likely to be involved in docking with NADPH-cytochrome P450 reductase, the structure of which has been obtained by the Kim and Masters laboratories, will be identified and subjected to module exchange or site-directed mutagenesis. 5) Fusion proteins containing one of the CYP4A subfamily members and NADPH- cytochrome P450 reductase and a module or modules from the constitutive nitric oxide synthases which mediate the Ca2+/calmodulin control of the latter enzymes will be constructed. Utilizing these approaches, the determinants of substrate specificities and catalytic efficiencies of the CYP4A enzymes will be determined.